SNU449 cells were transiently transfected with strep-tagged TM4SF5 11 (link) (or mock-transfected) for 24 h and then treated with Ab27 or Ab79 (10 µg/ml) for an additional 24 h. Cells were lysed in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2) containing 1% Brij58. Whole cell lysates (850 µg/condition) were immunoprecipitated with streptavidin beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4ºC for 16 h. The protein complexes were washed twice with ice-cold lysis buffer and then twice with PBS. The immunoprecipitated proteins were eluted by boiling in SDS sample buffer and analyzed by immunoblotting with anti-Strep tag (IBA; Olivette, MO, USA), anti-integrin α2 (Chemicon, Temecula, CA, USA), anti-integrin α5 (BD Biosciences), and anti-CD44 (BioLegend; San Diego, CA, USA) antibodies.
Anti integrin α5
Manufactured by BD
Sourced in United States
Anti-integrin α5 is a laboratory reagent that targets the α5 subunit of the integrin cell surface receptor. It is used in research applications to study the role of integrin α5 in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti integrin α2, by Merck Group (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti survivin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti sp1, by Santa Cruz Biotechnology (2 mentions)
Streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Anti cd44, by BioLegend (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
Anti upa, by Santa Cruz Biotechnology (1 mentions)
Imd 0354, by Merck Group (1 mentions)
Anti sp3, by Santa Cruz Biotechnology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti bmi 1, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti cyclin d2, by Cell Signaling Technology (1 mentions)
5 protocols using anti integrin α5
1
Immunoprecipitation of TM4SF5 Complexes
2
Analysis of Signaling Pathways in Cell Lysates
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Whole-cell lysates were prepared using RIPA buffer, as described previously15 (link), and analyzed using the following primary antibodies: anti-Sp1, anti-Sp3, anti-uPA, and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-myc (Upstate Biotechnology, Lake Placid, NY, USA); anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-ATF-2(T71), anti-ATF-2, anti-phospho-JNK(T183/Y185), anti-JNK, anti-phospho-Akt, anti-Akt, anti-survivin, anti-bcl-2, anti-phospho-IKKα/β(S176/180), anti-phospho-IκBα(S32), anti-phospho-NF-κB p65(S536), anti-cleaved caspase-3, and anti-PARP (Cell Signaling Tech., Danvers, MA, USA); anti-integrin α5 (BD Biosciences, San Jose, CA, USA); and anti-TMPRSS4 (in-house)15 (link). Where indicated, cells were treated with 0.1~10 μM KRT185314 (link) or IMD-0354 (Sigma, St Louis, MO, USA), 20~50 μM oxaliplatin (Sigma), or 0.1% dimethyl sulfoxide (DMSO) for 24~48 h before lysate preparation.
3
Whole-cell Lysates Preparation and Immunoblotting
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Whole-cell lysates were prepared using RIPA buffer as described previously [26 (link)] and analyzed using the following primary antibodies: anti-integrin α4, anti-SP1, and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX); anti-integrin α2 (Chemicon International, Temecula, CA); anti-myc (Upstate Biotechnology, Lake Placid, NY); anti-cyclin D2, anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-AKT, anti-AKT, anti-survivin, anti-Bcl-2, anti-SLUG, anti-SOX2, anti-CD133, anti-ABCB1, and anti-KLF4 (Cell Signaling Technology, Danvers, MA); anti-BMI1 (Millipore, Temecula, CA), anti-integrin α5 and anti-E-cadherin (BD Biosciences, San Jose, CA); anti-vimentin and anti-flag (Sigma); anti-TWIST1 (Abcam, Cambridge, MA); and anti-TMPRSS4 (in-house) [26 (link)].
4
Immunohistochemistry and Flow Cytometry Protocols
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We used the following primary antibodies: anti-FN (1:1,000, Millipore, Burlington MA USA), anti-α-SMA-Cy3 conjugated (1:500, Sigma-Aldrich, St. Louis, MO USA), anti-paxillin (1:300, BD Transduction, Spain), anti-keratin 6 (1:500, Covance, Spain), anti p-Smad2/3 (1:300, Thermo Fisher, Spain), anti-α5 integrin (1:100, Cell Signaling Technology, The Netherlands) and anti-Ki67 (1:200, Abcam, Cambridge, UK). For flow cytometry, we used anti-α5 integrin (1:200 BD Pharmingen, Spain) and anti β3 integrin (1:200 BD Bioscience, Spain). Secondary antibodies: anti-rabbit Alexa Fluor 488 (1:400 Thermo Fisher, Spain) and anti-mouse Alexa Fluor 647 (1:500, Invitrogen). To stain the cytoskeleton, we used Rhodamine Phalloidin (1:500 Thermo Fisher). To stain nuclei, we used either DAPI (1:10,000, Thermo Fisher) or Hoechst (1:10,000, Thermo Fisher).
5
Western Blot Analysis of Focal Adhesion Proteins
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Western blot analysis was performed as described previously20 (link). The antibodies used are: anti-FAK (3285S, Cell Signaling), anti-phospho-FAK (3283, Cell Signaling), ant-Flag (F1804, Sigma), anti-β1 integrin (ab179471, Abcam), anti-α5 integrin (553319, BD PharmingenTM), anti-Gal-1 (AF1245, R&D Systems), anti-GAPDH (ab8245, Abcam) and anti-β actin (GTX109639, Gene Tex).
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